Presently, there are numerous test devices available to simply and rapidly test liquids for the presence or absence of a particular analyte. For example, in regards to body fluids, tests are available to detect glucose, uric acid or protein in urine and to detect glucose, potassium ion or cholesterol in blood. Historically, most of the available diagnostic test devices have been sensitive to the volume of the test sample, as well as to the concentration of the particular analyte of interest.
The medical profession has provided an impetus to growth in this field by requiring analyte determinations that yield accurate and reproducible results and that can be performed quickly and cheaply. Such test methods are especially desirable in small or private medical offices, where fast and accurate results are required, but the volume of assay samples is low enough to preclude the investment in expensive diagnostic devices. In addition, the medical profession requires simple and essentially foolproof diagnostic tests, that are performable by relatively untrained personnel, due to the expense of having highly qualified personnel perform these routine assays.
As a result, several test methods have been developed that are inexpensive, fast, and easy to perform. Among the most widely used diagnostic devices are the "dip-and-read" type devices. These devices are widely used in the chemical analysis of biological fluids. For example, numerous physiological functions can be monitored merely by dipping a reagent strip device into a sample of body fluid, such as urine, and observing a detectable response, such as a change in color or a change in the amount of light reflected from or absorbed by the test device.
Many of the "dip-and-read" test devices for detecting body fluid components are capable of making quantitative or at least semiquantitative measurements. Thus, by measuring the response after a predetermined time, an analyst can obtain not only a positive indication of the presence of a particular analyte in a test sample, but also an estimate of how much of the analyte is present. Such test devices provide the physician with a facile diagnostic tool as well as the ability to gauge the extent of disease or of bodily malfunction.
Test devices such as these usually comprise one or more bibulous matrices, such as absorbent paper, having incorporated therein a particular test reagent that produces a detectable response, e.g., a color change, in the presence of a specific test sample analyte. Depending on the test reagent system incorporated into a particular bibulous matrix, the test devices can detect the presence of glucose, ketone bodies, cholesterol, triglycerides, bilirubin, urobilinogen, occult blood, nitrite, protein, urea, potassium, and other substances. A specific change in the intensity of color observed within a specific time range after contacting the test device with a sample, is indicative of the presence of a particular analyte and of the concentration of the analyte in the sample.
It is customary for reagent test devices to contain more than one test reagent-containing bibulous matrix, such that each test reagent-containing bibulous matrix is capable of detecting a particular analyte in a liquid test sample. For example, a diagnostic device could contain a test reagent-containing bibulous matrix responsive to glucose in urine and another bibulous matrix responsive to ketones, like acetoacetate, such that the second bibulous matrix is spaced from, but adjacent to, the glucose-responsive bibulous matrix. One diagnostic test device for urine contains eight adjacent test reagent-containing bibulous matrices providing analytical measurement of pH, protein, glucose, ketones, bilirubin, occult blood, nitrite, and urobilinogen.
For some assays, such as those performed on whole blood, it has been found that the normal method of simply dipping the diagnostic device into the liquid sample cannot be used. For such assays the amount or volume of the test sample contacting the test-reagent containing diagnostic device is very critical. For example, dry reagent methods for testing whole blood or serum require the application of specific test sample volumes and the use of sophisticated filtering and separating techniques to obtain accurate results.
Therefore, in order to achieve accurate and reproducible results, a very precise amount of sample must contact the test-reagent containing bibulous matrix each time an assay is performed. For these assays in particular, the development of a volume independent diagnostic device, wherein a precise and reproducible amount of test sample contacts the test reagent-containing bibulous matrix each time an assay is performed, would be extremely advantageous. Such a device would overcome the problems of inaccurate and inconsistent results due to differences in the amount of test sample contacting the test reagent in the bibulous matrix. The method and device of the present invention is primarily directed at providing a constant sample loading of test sample per unit volume of a test reagent-containing bibulous matrix, and as a result, a truly volume independent diagnostic device.
Other considerations also arise in developing a process and device for testing liquids for a specific analyte. One important consideration is the gross sample size needed to perform the analyte determination. For instance, in testing whole blood, an ideal process includes withdrawing a whole blood sample in "noninvasive" amounts, such as a pin prick drop, and immediately depositing the undiluted whole blood sample on the diagnostic device.
Another consideration is the degree of sophistication of the technician performing the assay. It is often desirable to have relatively untrained personnel carry out routine assays and obtain accurate quantitative results. In these situations, it is important that the assay method contain a minimum of manipulative steps, be free of possible interferences or contamination and provide for easy measurement. For instance, among the several possible manipulative steps, testing the incorrect sample or applying the incorrect amount of sample to the diagnostic device are the most probable areas of assay error.
Therefore, a need exists for a process and device for rapidly and accurately testing a small volume of liquid for a particular analyte, wherein accurate and reproducible analyte concentrations are obtained independent of sample size. Such a method and device for determining analyte concentrations in liquids would allow medical personnel to carry out analyte assays on a more routine and more confident basis.
The "dip-and-read" method for testing urine samples has enjoyed great success due to the ease, speed and low cost of testing liquid samples. However, substantial work is still being performed in this area as diagnostic device uses are demanding more accurate test methods, for more analytes, on smaller liquid test samples. Diagnostic device users are especially eager to reduce the possibility of test inaccuracy, usually by making the test method simpler and less operator dependent. The ideal way to reduce operator dependence is to eliminate the need to dilute the liquid test sample and to eliminate the need to introduce a precise sample volume to the diagnostic device. It is to the latter objective that the method and device of the present invention is directed.
Indicative of the work conducted in this field is U.S. Pat. No. 3,798,004 to Zerachia et al, disclosing a semiquantitative method for determining analyte concentrations with a laminated device including a reagent-impregnated matrix placed between a pair of liquid impervious members. The analyte-containing sample contacts the reagent-impregnated matrix along the matrix-exposed edge of the test device. As the analyte-containing sample progresses inwardly towards the center of the matrix, the analyte reacts with the reagent impregnated in the matrix to provide a visible color pattern. The depth of the inward penetration of the color pattern is measured to determine the concentration of the analyte in the test solution. The amount of test sample absorbed by the matrix is limited by the capacity of the matrix to hold liquids, so a semiquantitative determination of an analyte is possible.
Similarly, Morison in U.S. Pat. No. 3,620,677 describes an indicating device including an impervious material encasing a reagent-treated capillary material, such that at least some of the capillary material is exposed. The analyte-containing sample is applied to the capillary material, and, as the sample chromatographs through the reagent-treated capillary material, a chromogenic reaction occurs. After complete analyte reaction, the chromogenic reaction ceases. Therefore the point that the color formation ends gives a reading of the approximate analyte concentration. The method disclosed in the Morison patent is volume dependent, as the amount of analyte, and therefore the degree of the chromogenic reaction, increases with sample volume.
Nussbaum in U.S. Pat. No. 3,810,739 discloses a reagent-impregnated paper encased in a plastic covering, so arranged such that a test sample can be introduced only through a single opening. A chromogenic reaction occurs within the device, and is observed through the translucent plastic used to encase the reagent-impregnated paper. The method and device of the Nussbaum patent are directed to qualitatively determining the presence or absence of a particular chemical or bacterial constituent of a solid or liquid sample. The device is constructed to retard sample evaporation during periods of long reaction incubation, especially at high temperatures. As a result, the volume or weight of test sample is an unimportant variable in the method disclosed in the Nussbaum patent.
U.S. Pat. No. 4,069,017 to Wu et al discloses contacting adjacent matrices in order to provide uniform distribution of a test sample from a first, untreated matrix to a second, reagent-impregnated matrix. Although the device does reduce volume dependence, the configuration of the matrices is specifically designed to provide a uniform bilirubin distribution to the reagent-impregnated matrix for uniform binding and chromogenic reaction.
Kondo et al U.S. Pat. No. 4,256,693 discloses a multilayered device including a layer to remove insoluble constituents, a waterproof layer with an opening, a porous spreading layer and a reagent layer, in that order. This device delivers a less-than-saturating volume of test sample from the spreading layer to the reagent layer as a method to ensure even sample distribution. Accordingly, the spreading layer does not and could not act as a barrier layer.
U.K. Pat. No. 2,090,549 discloses an analytical device for metering a precise quantity of blood utilizing a metering channel. Capillary action draws only a certain amount of blood into the metering channel for distribution through a filter layer to a layer impregnated with a reagent.
U.S. Pat. No. 4,647,430 discloses a volume independent test device wherein a reagent-impregnated matrix is completely covered by a microporous film. A liquid sample penetrates the film until the matrix is saturated, resulting in a constant loading of sample per unit area.